Elucidation of the mechanism of aggregation of disease-associated single point mutants of the murine prion protein on the basis of time-resolved 2D NMR spectroscopy in combination with complementary kinetic methods

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The aggregation mechanism of the prion protein is known to be complex. It is highly heterogeneous since multiple oligomeric as well as fibrillar structures are being populated. It is essential to understand the disease-associated assembly of prion protein monomers into high-molecular-weight polymers in mechanistic terms in order to be able to develop tailored therapeutic approaches. This requires following the aggregation process in detail on the molecular level. Kinetic methods are especially suited in this respect as the desired mechanistic information is contained in the kinetic data.We have investigated the aggregation of the wildtype murine prion protein using kinetic two-dimensional NMR spectroscopy. By following the decrease in the monomer concentration we have been able to delineate heterogeneity in the aggregation kinetics on the basis of amino-acid-specific differences. Within the scope of the proposed project we aim at extending our kinetic studies and exploiting the unique structural resolution of NMR which cannot be attained using other methods. Apart from measuring the kinetics of the decrease in the monomer concentration we specifically plan to characterize the kinetics of distinct oligomeric populations on the path to amyloid-like fibrils using 2D NMR methods. In order to so, we will make use of methods for isotope-specific labelling of methyl groups in combination with methyl TROSY NMR experiments. Complementary kinetic data on oligomeric as well as fibrillar populations will be collected using atomic force microscopy, SDS PAGE, fluorescence and absorption spectroscopies. In the end, the kinetic data from the different experiments will be globally fitted in order to derive a model for the aggregation of the prion protein which is most compatible with the experimental data. We will then characterize the aggregation kinetics of disease-associated single point mutants of the prion protein and thereby identify the aggregation sub-steps affected by the different mutations.